This invention relates to novel macrolide compounds that are useful as antibacterial and antiprotozoal agents in mammals, including man, as well as in fish and birds. This invention also relates to pharmaceutical compositions containing the novel compounds and to methods of treating bacterial and protozoal infections in mammals, fish and birds by administering the novel compounds to mammals, fish and birds requiring such treatment.
Macrolide antibiotics are known to be useful in the treatment of a broad spectrum of bacterial and protozoal infections in mammals, fish and birds. Such antibiotics include various derivatives of erythromycin A such as azithromycin which is commercially available and is referred to in U.S. Pat. Nos. 4,474,768 and 4,517,359, both of which are incorporated herein by reference in their entirety. Other macrolide antibiotics are disclosed and claimed in PCT international application number PCT/IB98/00741, filed May 15, 1998 (Attorney docket number PC 9726A), which designates the United States, and U.S. Pat. No. 5,527,780, issued Jun. 18, 1996. U.S. Pat. No. 5,527,780 and PCT international application number PCT/IB98/00741 are incorporated herein by reference in their entirety. Like azithromycin and other macrolide antibiotics, the novel macrolide compounds of the present invention possess activity against various bacterial and protozoal infections as described below.
The present invention relates to compounds of the formula 
and to pharmaceutically acceptable salts, prodrugs and solvates thereof, wherein:
X1 is xe2x80x94CH2xe2x80x94 or xe2x80x94NR4xe2x80x94;
X2 is xe2x95x90O or xe2x95x90NOR1;
Z is H, C1-C14 alkyl, (C6-C10 aryl)(C1-C10 alkyl)- or (4-10 membered heterocyclic)(C1-C10 alkyl)-, wherein one or two carbon atoms of the foregoing alkyl moieties are optionally replaced by a heteroatom selected from O, S and xe2x80x94N(R4), and the foregoing groups, except H, are optionally substituted with 1 to 3 substituents independently selected from halo, hydroxy, C1-C14 alkoxy, C1-C14 alkyl, (C6-C10 aryl)(C1-C10 alkoxy)- and (4-10 membered heterocyclic)(C1-C10 alkoxy)-;
R1 is H, methyl or ethyl;
R2 is a group of the formula 
wherein n is an integer from 1 to 4;
R3 is C6-C10 aryl or 4-10 membered heterocyclic, wherein said R3 is optionally substituted by I to 3 substituents independently selected from the group consisting of C1-C6 alkoxy, trifluoromethyl, trifluoromethoxy, halo, and xe2x80x94NR4R5;
each R4 and R5 is independently selected from H and C1-C6 alkyl;
R6 is H; and,
R7 and R8 are each independently selected from H and C1-C6 alkyl except that at least one of R7 and R8 is C1-C6 alkyl.
Preferred compounds of formula 1 include those wherein Z is H, X1 is xe2x80x94NHxe2x80x94 or xe2x80x94CH2xe2x80x94, n is 2, R7 is C1-C3 alkyl, R8 is H or C1-C3 alkyl, X2 is O, xe2x95x90NOCH3 or xe2x95x90NOCH2CH3, and R3 is 5 or 6-membered aromatic heterocyclic containing 1 or 2 nitrogen atoms in said heterocyclic ring. More preferred are the foregoing compounds wherein n is 2, R7 is methyl or ethyl, R8 is H, methyl or ethyl, and R3 is pyridyl.
Specific preferred compounds of formula 1 include those having the structure of formula 33 
and pharmaceutically acceptable salts, prodrugs and solvates thereof; wherein X1 is NH or xe2x80x94CH2xe2x80x94; X2 is xe2x95x90O or xe2x95x90NOR1; and R1 is H, methyl or ethyl. More preferred compounds are those compounds of formula 33 wherein x2 is O, xe2x95x90NOCH3 or xe2x95x90NOCH2CH3.
Other specific preferred compounds of formula 1 include those having the structure of formula 32 
and pharmaceutically acceptable salts, prodrugs and solvates thereof; wherein X1 is NH or xe2x80x94CH2xe2x80x94; X2 is xe2x95x90O or xe2x95x90NOR1; and R1 is H, methyl or ethyl. More preferred compounds are those compounds of formula 32 wherein X2 is O, xe2x95x90NOCH3 or xe2x95x90NOCH2CH3.
The present invention also relates to the preparation of compounds of formula 30 
which comprises treating a compound of formula 19 with a compound of formula 29 
wherein X2, R7, R3 and R8 are as defined above, in a solvent, preferably toluene.
The present invention also relates to intermediates of the formula 29 
wherein R3, R7 and R8 are as defined above.
The invention also relates to a pharmaceutical composition for the treatment of a bacterial infection or a protozoa infection, or a disorder related to a bacterial or protozoal infection, in a mammal, fish, or bird, which comprises a therapeutically effective amount of a compound of formula 1, or a pharmaceutically acceptable salt, prodrug or solvate thereof, and a pharmaceutically acceptable carrier.
The invention also relates to a method of treating a bacterial infection or a protozoa infection, or a disorder related to a bacterial or protozoal infection, in a mammal, fish, or bird which comprises administering to said mammal, fish or bird a therapeutically effective amount of a compound of formula 1 or a pharmaceutically acceptable salt, prodrug or solvate thereof.
The invention also relates to a method of treating cancer or atherosclerosis in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula 1 or a pharmaceutically acceptable salt, prodrug or solvate thereof.
The term xe2x80x9ctreatingxe2x80x9d, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term xe2x80x9ctreatmentxe2x80x9d, as used herein, refers to the act of treating, as xe2x80x9ctreatingxe2x80x9d is defined immediately above.
As used herein, unless otherwise indicated, the terms or phrases xe2x80x9cbacterial infection(s)xe2x80x9d, xe2x80x9cprotozoal infection(s)xe2x80x9d, and xe2x80x9cdisorders related to bacterial infections or protozoal infectionsxe2x80x9d include the following: pneumonia, otitis media, sinusitus, bronchitis, tonsillitis, and mastoiditis related to infection by Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Enterococcus faecalls, E. faecium, E. casselflavus, S. epidermidis, S. haemolyticus, or Peptostreptococcus spp.; pharyngitis, rheumatic fever, and glomewulonephritis related to infection by Streptococcus pyogenes, Groups C and G streptococci, Corynebacterium diphtheriae, or Actinobacillus haemolyticum; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae; blood and tissue infections, including endocarditis and osteomyelitis, caused by S. aureus, S. haemolyticus, E faecalis, E faecium, E. durans, including strains resistant to known antibacterials such as, but not limited to, beta-lactams, vancomycin, aminoglycosides, quinolones, chloramphenicol, tetracylines and macrolides; uncomplicated skin and soft tissue infections and abscesses, and puerperal fever related to infection by Staphylococcus aureus, coagulase-negative staphylococci (i.e., S. epidermidis, S. hemolyticus, etc.), Streptococcus pyogenes, Streptococcus agalacfiae, Streptococcal groups C-F (minute-colony streptococci), viridans streptococci, Corynebactetium minutissimum, Clostridium spp., or Bartonella henselae; uncomplicated acute urinary tract infections related to infection by Staphylococcus aureus, coagulase-negative staphylococcal species, or Enterococcus spp.; urethritis and cervicitis; sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducreyi, Treponema pallidum, Ureaplasma urealyticum, or Neiserria gonorrheae; toxin diseases related to infection by S. aureus (food poisoning and toxic shock syndrome), or Groups A, B, and C streptococci; ulcers related to infection by Helicobacter pylori; systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, and dacrocystitis related to infection by Chlamydia trachomatis, Neissena gonorrhoeae, S. aureus, S. pneumoniae, S. pyogenes, H. influenzae, or Listenta spp.; disseminated Mycobacterium avium complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellulare; infections caused by Mycobacterium tuberculosis, M leprae, M. paratuberculosis, M. kansasii, or M. chelonei; gastroenteritis related to infection by Campylobacter jejuni; intestinal protozoa related to infection by Cryptosporidium spp.; odontogenic infection related to infection by viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related to infection by Clostridium perfringens or Bacteroides spp.; and atherosclerosis or cardiovascular disease related to infection by Helicobacter pylori or Chiamydia pneumoniae. Bacterial infections and protozoal infections, and disorders related to such infections, which may be treated or prevented in animals include the following: bovine respiratory disease related to infection by P. haemolytica, P. multocida, Mycoplasma bovis, or Bordetelia spp.; cow enteric disease related to infection by E coli or protozoa (i.e., coccidia, cryptosporidia, etc.); dairy cow mastitis related to infection by S. aureus, Strep. uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Klebsiella spp., Corynebactefium, or Enterococcus spp.; swine respiratory disease related to infection by A. pleuro., P. multocida, or Mycoplasma spp.; swine enteric disease related to infection by E coli Lawsonia intracellulans, Salmonella, or Serpulina hyodysintenae; cow footrot related to infection by Fusobacterium spp.; cow metritis related to infection by E coli; cow hairy warts related to infection by Fusobacterium necrophorum or Bacteroides nodosus; cow pinkye related to infection by Moraxelia bovis; cow premature abortion related to infection by protozoa (i.e. neosporium); urinary tract infection in dogs and cats related to infection by E coli; skin and soft tissue infections in dogs and cats related to infection by S. epidermidis, S. intermedius, coagulase neg. Staphylococcus or P. multocida; and dental or mouth infections in dogs and cats related to infection by Alcaligenes spp., Bacteroides spp., Clostridium spp., Enterobacter spp., Eubactenrum, Peptostreptococcus, Porphyromonas, or Prevotella. Other bacterial infections and protozoal infections, and disorders related to such infections, which may be treated or prevented in accord with the method of the present invention are referred to in J. P. Sanford et al., xe2x80x9cThe Sanford Guide To Antimicrobial Therapy,xe2x80x9d 26th Edition, (Antimicrobial Therapy, Inc., 1996).
The term xe2x80x9chaloxe2x80x9d, as used herein, unless otherwise indicated, includes fluoro, chloro, brorno or iodo. Preferred halo groups are fluoro, chloro and bromo.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight or branched moieties. Said alkyl group may include one or two double or triple bonds. It is understood that for said alkyl group to include a carboncarbon double or triple bond at least two carbon atoms are required in said alkyl group.
The term xe2x80x9carylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl.
The term xe2x80x9c4-10 membered heterocyclicxe2x80x9d, as used herein, unless otherwise indicated, includes aromatic and non-aromatic heterocydic groups containing one or more heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 4-10 atoms in its ring system. Non-aromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or more oxo moieties. An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5 membered heterocyclic group is thiazolyl and an example of a 10 membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthynidinyl, and furopyridinyl. The foregoing groups, as derived from the compounds listed above, may be C-attached or N-attached where such is possible. For instance, a group derived from pyrrole may be pyrrol-1-yl (N-attached) or pyrrol-3yl (C-attached).
As used herein, unless otherwise indicated, xe2x80x9cAcxe2x80x9d indicates an acetyl group.
As used herein, unless otherwise indicated, xe2x80x9cMexe2x80x9d indicates a methyl group.
As used herein, unless otherwise indicated, xe2x80x9cETxe2x80x9d indicates an ethyl group.
The phrase xe2x80x9cpharmaceutically acceptable salt(s)xe2x80x9d, as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compoun ds of the present invention. The compounds of the present invention that are basic in nature are c ap able of forming a wid e variety of s alts with various inorganic and organic a cids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds of are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, ptoluenesulfonate and pamoate [I.E., 1,1xe2x80x2-methylene-bis-(2-hydroxy-3-naphthoate)] salts. The compounds of the present invention that include a basic moiety, such as an amino group, may formn pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
Those compounds of the present invention that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkaline earth metal salts and, particularly, the calcium, magnesium, sodium and potassium salts of the compounds of the present invention.
The subject invention also includes isotopically-labelled compounds, and the pharmaceutically acceptable salts thereof, which are identical to those recited in formula 1, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, and 36Cl, respectively. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds of formula 1 of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples and Preparations below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.
This invention also encompasses pharmaceutical compositions containing and methods of treating bacterial infections through administering prodrugs of compounds of the formula 1. Compounds of formula 1 having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of formula 1. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, omithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. The amide and ester moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Free hydroxy groups may be derivatized using groups induding but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in D. Fleisher, R. Bong, B. H. Stewart, Advanced Drug Delivery Reviews (1996) 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in R. P. Robinson et al., J. Medicinal Chemistry (1996) 39, 10.
Certain compounds of formula 1 may have asymmetric centers and therefore exist in different enantiomeric forms. This invention relates to the use of all optical isomers and stereoisomers of the compounds of formula 1 and mixtures thereof. In particular, the invention includes both the R and S configurations of the methyl group at C-10 of the macrolide ring of formula 1, and both the E and Z isomers of the xe2x80x94OR1 group connected to the nitrogen of the oxime moiety at C-9 of the macrolide ring of formula 1.
The preparation of the compounds of the present invention is illustrated in the following Schemes. 
The preparation of the compounds of the present invention is illustrated in the above schemes. The synthesis of racemic as well as enantiomerically pure side chains is illustrated in Schemes 1 and 2. In Scheme 1, the compound of formula 2, in which R7 is an alkyl group as defined above, is commercially available or may be prepared according to methods familiar to those skilled in the art Protection of the primary hydroxyl group as its t-butyidimethylsilyl ether (represented as OTBS in the compound of formula 3) may be done by treatment of the compound of formula 2 with 1 equivalent of t-butyldimethylsilyl chloride and imidazole in N,N-dimethylformamide (DMF) at room temperature (approximately 20-25xc2x0 C). Conversion of the secondary alcohol to the corresponding mesylate of formula 3 (in which Ms represents the mesylate moiety) may be done by reaction with methanesulfonyl chloride and triethylamine in dichloromethane at approximately xe2x88x9220xc2x0 C. Displacement of the mesylate with the compound of formula 4, wherein R3 is as defined above, to provide the compound of formula 5 may be accomplished by reaction of the compound of formula 4 with a base such as sodium hydride or potassium carbonate at approximately 80xc2x0 C. followed by addition of the mesylate compound of formula 3. The compound of formula 4 may be prepared according to methods familiar to those skilled in the art, including one or more synthetic methods described in H. Bredereck, R. Gompper, H. G. v. Schuh, and G. Theilig, Angew. Chem., 24, 753 (1959). Deprotection of the silylether and conversion of the resultant alcohol to the amine of formula 6 may be achieved by the sequence: (1) treatment of the compound of formula 5 with tetrabutylammonium fluoride in tetrahydrofuran (THF) to provide the corresponding alcohol, (2) reaction of the alcohol with methanesulfonyl chloride and triethylamine to produce the mesylate, (3) displacement of the mesylate with sodium azide in DMF at room temperature to yield the primary azide, and (4) hydrogenation of the azide over palladium on carbon in methanol to provide the primary amine of formula 6. This compound may be introduced into the macrolide structure as the side chain represented as xe2x80x94X1xe2x80x94R2 in the compound of formula 1 according to the methods described herein and according to one or more methods described in U.S. Pat. No. 5,527,780 and PCT international application number PCT/IB98/00741, referred to above. Analogous side chains represented as xe2x80x94X1xe2x80x94R2 in the compound of formula 1 may be prepared in a similar manner.
While the side chain compounds prepared according to Scheme 1 are racemic, those prepared according to Scheme 2 are substantially enantiomerically pure. With reference to Scheme 2, the enantiomerically pure compound of the formula 7, which is commercially available, such as Sxe2x80x94(-)-methyl lactate, or prepared according to methods familiar to those skilled in the art, may be converted to its t-butyidimethylsilyl ether by treatment with t-butyidimethylchlorosilane in DMF in the presence of imidazole at a temperature ranging from 0xc2x0 C. to 40xc2x0 C., preferably at room temperature. Reduction of this compound with di-isobutylaluminum hydride in toluene at approximately xe2x88x9270xc2x0 C. provides the aldehyde of formula 8 (wherein TBS represents t-butyldimethylsilyl). Wittig coupling of the compound of formula 8 with carbethoxymethylene triphenylphosphorane in benzene at a temperature ranging from 60xc2x0 C. to 80xc2x0 C. produces the corresponding unsaturated ester which may be hydrogenated over palladium in ethyl acetate to provide the ester of formula 9. Reduction of the ester to the corresponding alcohol using lithium aluminum hydride in THF, conversion of the alcohol to the corresponding mesylate by treatment with methanesulfonyl chloride and triethylamine in dichloromethane at a temperature ranging from xe2x88x9220xc2x0 C. to 0xc2x0 C., and, finally, displacement of the mesyl group with azide by reaction with sodium azide in DMF at room temperature affords the azide of formula 10. Hydrogenation of the compound of formula 10 over palladium in a polar solvent, such as methanol, followed by reaction with benzylchloroformate provides the benzyloxycarbonyl amide of formula 11 (wherein Cbz represents benzyloxycarbonyl). Desilylation with tetra-n-butylammonium fluoride in THF followed by treatment with methanesulfonyl chloride and triethylamine at a temperature ranging from xe2x88x9220xc2x0 C. to 0xc2x0 C. produces the corresponding mesylate of formula 11A (wherein Ms represents methanesulfonyl). Reaction of the compound of formula 11A with a compound of the formula 4 (which is illustrated in Scheme 1) and sodium hydride in dry DMF at a temperature ranging from 20xc2x0 C. to 100xc2x0 C., followed by deprotection by hydrogenation over a palladium catalyst in methanol at room temperature, provides a compound of the formula 12. An example of a compound corresponding to formula 12 is (R)-4-(4-pyridin-3-yl-imidazol-1-yl)-pentylamine. Following the same procedures outlined above except starting with a compound having the opposite stereochemical orientation with respect to the hydroxy group, such as R-(+)-methyl lactate, provides a compound corresponding to the compound of formula 12 except the stereochemical orientation of the R7 group is opposite to that illustrated for the compound of formula 12. An example of such a compound is (S)-4-(4-pyridin-3-yl-imidazol-1-yl)-pentylamine.
The synthesis of the final ketolide is illustrated in Scheme 3. The compound of formula 13, wherein R6 is acetyl, may be prepared as described in U.S. Pat. No. 5,543,400 (issued Aug. 6, 1996). In general, the intermediate compound of formula 14 may be prepared as described in U.S. Pat. No. 5,543,400, PCT international application number PCT/IB98/00741 and U.S. Pat. No. 5,527,780, each of which is referred to above, and also United Kingdom patent application number 2,288,174 (published Oct. 11, 1995), and G. Griesgraber et al., xe2x80x9c3-Keto-11,12-carbazate Derivatives of 6O-Methylerythromycin A,xe2x80x9d Journal of Antibiotics, 49(5), 465-477 (1996).
In step 1 of Scheme 3, compounds of the formula 14 wherein R6 is H, X1 is xe2x80x94CH2xe2x80x94 and R2 and R3 are as defined above, may be prepared by treating a compound of the formula 13 with a compound of the formula H2Nxe2x80x94X1xe2x80x94R2, wherein X1 is xe2x80x94CH2xe2x80x94 and R2 is as defined above, in a solvent such as acetonitrile, DMF, THF, dimethoxy ethane or dimethylsulfoxide (DMSO), preferably acetonitrile, at a temperature within the range of about 50xc2x0 C. to 90xc2x0 C., preferably about 80xc2x0 C., for a period of about 4 to 16 hours. Compounds of the formula 14, wherein X1 is xe2x80x94NHxe2x80x94 and R2 is as defined above, can be prepared as described below in reference to schemes 4-6 and further as described in United Kngdom patent application number 2,288,174, referred to above. In step 2 of Scheme 3, compounds of the formula 15 may be prepared by treating a compound of the formula 14 with a compound of the formula R1ONH2xe2x80xa2HCl or R1ONH2, wherein R1 is as defined above, in the presence of an acid, such as Pyxe2x80xa2HCl (wherein Py denotes pyridine) or Et3Nxe2x80xa2HCl (wherein Et denotes ethyl), in a polar solvent, preferably methanol, ethanol, or isopropyl alcohol, at a temperature within the range of about 65xc2x0 C. to 95xc2x0 C. for a period of about 10 hours to 6 days.
Scheme 4 illustrates the preparation of compounds of formula 1 wherein X1 is xe2x80x94NHxe2x80x94 and R2 is as defined above. In particular, Scheme 4 illustrates an R2 moiety wherein xe2x80x9cnxe2x80x9d is 2, although groups wherein xe2x80x9cnxe2x80x9d has other values may be used following an analogous procedure. In the compounds illustrated in Scheme 4, R7 is an alkyl group and R8 (not shown) is H. In step 1 of Scheme 4, a compound of formula 16 is treated with a compound of formula 4 in THF at room temperature to provide the compound of formula 17. Reduction with di-isobutylaluminum hydride in dichloromethane at approximately xe2x88x9270xc2x0 C. provides the aldehyde of formula 18. The compound of formula 19, wherein X2 is as defined above, may be prepared as described in PCT international application number PCT/IB98/00741 and U.S. Pat. No. 5,527,780, referred to above. Further, the synthesis of 11,12-cyclic carbazates analogous to the compounds of formula 19 is described in W. R. Baker, J. D. Clark, R. L. Stephens, and K. H. Kim, J. Org. Chem., 53, 2340 (1988). Condensation of the aldehyde of formula 18 with the compound of formula 19 in toluene at approximately 100xc2x0 C. followed by reduction of the resultant imine in methanol with sodium cyanoborohydride at 23xc2x0 C. gave rise to the compound of formula 20 wherein the product is racemic with respect to the chiral carbon to which R7 is attached.
Scheme 5 illustrates the preparation of compounds that are similar to those of formula 20 except the product is substantially enantiomerically pure with respect to the chiral carbon to which R7 is attached. This is indicated in structures 21-26 in Scheme 5 wherein the asterisk represents a specific stereoisomeric orientation (specifically, R or S) with respect to the carbon to which R7 is attached. In the compounds illustrated in Scheme 5, R7 is an alkyl group and R8 (not shown) is H. The synthesis of these compounds begins with chiral starting materials, which are illustrated here as R- or S-1,3-butanediol (the compound of formula 21). Mono-silylation by reaction with 1 equivalent of t-butyidimethylsilyl chloride in DMF in the presence of imidazole at room temperature, about 23xc2x0 C., for about 12 hours provides mono-silyl ether (the compound of formula 22 wherein TBS is t-butyidimethylsilyl). Mesylation by treatment with 1 equivalent of methanesulfonyl chloride and triethylamine in dichloromethane at approximately xe2x88x9220xc2x0 C. for approximately 40 minutes provides the corresponding mesylate of formula 23 (wherein Ms denotes the mesylate moiety). Displacement of the mesylate with a compound of formula 4 (illustrated in Scheme 1) in DMF with sodium hydride provides the compound of formula 24 with complete inversion of the stereochemistry of the alpha R7 group. Desilylation by reaction with tetra-t-butylammonium fluoride in THF followed by swem oxidation with oxalyl chloride and DMSO provides the chiral alpha-R7 imidazole propionaldehyde of formula 25. Coupling of the aldehyde with the cyclic carbazate of formula 19, as described in reference to Scheme 4 above, provides the R-R7 or Sxe2x80x94R7 compound of formula 26.
Scheme 6 illustrates the preparation of compounds of formula 26 which are similar to those of formula 20 except that both R7 and R8 are alkyl groups. The synthesis illustrated in Scheme 6 follows the same general steps and conditions of the synthesis illustrated in Scheme 4. The starting materials are either commercially available or they may be prepared according to synthetic methods familiar to those skilled in the art.
The compounds of the present invention may have asymmetric carbon atoms. Such diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known to those skilled in the art, for example, by chromatography or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixtures into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomeric mixtures and pure enantiomers are considered as part of the invention.
The compounds of formula 1 that are basic in nature are capable of forming a wide variety of different salts with various inorganic and organic acids. Although such salts must be pharmaceutically acceptable for administration to animals, it is often desirable in practice to initially isolate the compound of formula 1 from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent and subsequently convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds of this invention are readily prepared by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is readily obtained. The desired acid salt can also be precipitated from a solution of the free base in an organic solvent by adding to the solution an appropriate mineral or organic acid.
Those compounds of the formula 1 that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include the alkali metal or alkalineearth metal salts and particularly, the sodium and potassium salts. These salts may be prepared by conventional techniques. The chemical bases which are used as reagents to prepare the pharmaceutically acceptable base salts of this invention are those which form non-toxic base salts with the acidic compounds of formula 1. Such non-toxic base salts include those derived from such pharmacologically acceptable cations as sodium, potassium calcium and magnesium, etc. These salts can be prepared by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations, and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they may also be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric quantities of reagents are preferably employed in order to ensure completeness of reaction and maximum yields of the desired final product.
The activity of the compounds of the present invention against bacterial and protozoa pathogens is demonstrated by the compound""s ability to inhibit growth of defined strains of human (Assay I) or animal (Assays II and ll) pathogens.
Assay I, described below, employs conventional methodology and interpretation criteria and is designed to provide direction for chemical modifications that may lead to compounds that circumvent defined mechanisms of macrolide resistance. In Assay I, a panel of bacterial strains is assembled to include a variety of target pathogenic species, including representatives of macrolide resistance mechanisms that have been characterized. Use of this panel enables the chemical structure/activity relationship to be determined with respect to potency, spectrum of activity, and structural elements or modifications that may be necessary to obviate resistance mechanisms. Bacterial pathogens that comprise the screening panel are shown in the table below. In many cases, both the macrolide-susceptible parent strain and the macrolide-resistant strain derived from it are available to provide a more accurate assessment of the compound""s ability to circumvent the resistance mechanism. Strains that contain the gene with the designation of ermA/ermB/ermC are resistant to macrolides, lincosamides, and streptogramin B antibiotics due to modifications (methylation) of 23S rRNA molecules by an Erm methylase, thereby generally prevent the binding of all three structural classes. Two types of macrolide efflux have been described; msrA encodes a component of an efflux system in staphylococci that prevents the entry of macrolides and streptogramins while mefA/E encodes a transmembrane protein that appears to effiux only macrolides. Inactivation of macrolide antibiotics can occur and can be mediated by either a phosphorylation of the 2xe2x80x2-hydroxyl (mph) or by cleavage of the macrocyclic lactone (esterase). The strains may be characterized using conventional polymerase chain reaction (PCR) technology and/or by sequencing the resistance determinant. The use of PCR technology in this application is described in J. Sutcliffe et al., xe2x80x9cDetection Of Erythromycin-Resistant Determinants By PCRxe2x80x9d, Antimicrobial Agents and Chemotherapy, 40(11), 2562-2566 (1996). The assay is performed in microtiter trays and interpreted according to Performance Standards for Antimicrobial Disk Susceptibility Testsxe2x80x94Sixth Edition; Approved Standard, published by The National Committee for Clinical Laboratory Standards (NCCLS) guidelines; the minimum inhibitory concentration (MIC) is used to compare strains. Compounds are initially dissolved in dimethylsulfoxide (DMSO) as 40 mg/ml stock solutions.
Assay II is utilized to test for activity against Pasteurella multocida and Assay III is utilized to test for activity against Pasteurella haemoblyca. 
This assay is based on the liquid dilution method in microliter format. A single colony of P. multocida (strain 59A067) is inoculated into 5 ml of brain heart infusion (BHI) broth. The test compounds are prepared by solubilizing 1 mg of the compound in 125 xcexcl of dimethylsulfoxide (DMSO). Dilutions of the test compound are prepared using uninoculated BHI broth. The concentrations of the test compound used range from 200 xcexcg/ml to 0.098 xcexcg/ml by twofold serial dilutions. The P. multocida inoculated BHI is diluted with uninoculated BHI broth to make a 104 cell suspension per 200 xcexcl. The BHI cell suspensions are mixed with respective serial dilutions of the test compound, and incubated at 37xc2x0 C. for 18 hours. The minimum inhibitory concentration (MIC) is equal to the concentration of the compound exhibiting 100% inhibition of growth of P. multocida as determined by comparison with an uninoculated control.
This assay is based on the agar dilution method using a Steers Replicator. Two to five colonies isolated from an agar plate are inoculated into BHI broth and incubated overnight at 37xc2x0 C. with shaking (200 rpm). The next morning, 300 xcexclof the fully grown P. haemolytfca preculture is inoculated into 3 ml of fresh BHI broth and is incubated at 37xc2x0 C. with shaking (200 rpm). The appropriate amounts of the test compounds are dissolved in ethanol and a series of two-fold serial dilutions are prepared. Two ml of the respective serial dilution is mixed with 18 ml of molten BHI agar and solidified. When the inoculated P. haemolytica culture reaches 0.5 McFarland standard density, about 5 xcexcl of the P. haemolytca culture is inoculated onto BHI agar plates containing the various concentrations of the test compound using a Steers Replicator and incubated for 18 hours at 37xc2x0 C. Initial concentrations of the test compound range from 100-200 xcexcg/ml. The MIC is equal to the concentration of the test compound exhibiting 100% inhibition of growth of P. haemolytica as determined by comparison with an uninoculated control.
The in vivo activity of the compounds of formula 1 can be determined by conventional animal protection studies well known to those skilled in the art, usually carried out in mice.
Mice are allotted to cages (10 per cage) upon their arrival, and allowed to acclimate for a minimum of 48 hours before being used. Animals are inoculated with 0.5 ml of a 3xc3x97103 CFU/ml bacterial suspension (P. multocida strain 59A006) intraperitoneally. Each experiment has at least 3 non-medicated control groups including one infected with 0.1X challenge dose and two infected with 1X challenge dose; a 10X challenge data group may also be used. Generally, all mice in a given study can be challenged within 30-90 minutes, especially if a repeating syringe (such as a Cornwall(copyright) syringe) is used to administer the challenge. Thirty minutes after challenging has begun, the first compound treatment is given. It may be necessary for a second person to begin compound dosing if all of the animals have not been challenged at the end of 30 minutes. The routes of administration are subcutaneous or oral doses. Subcutaneous doses are administered into the loose skin in the back of the neck whereas oral doses are given by means of a feeding needle. In both cases, a volume of 0.2 ml is used per mouse. Compounds are administered 30 minutes, 4 hours, and 24 hours after challenge. A control compound of known efficacy administered by the same route is included in each test Animals are observed daily, and the number of survivors in each group is recorded. The P. multocida model monitoring continues for 96 hours (four days) post challenge.
The PD50 is a calculated dose at which the compound tested protects 50% of a group of mice from mortality due to the bacterial infection which would be lethal in the absence of drug treatment.
The compounds of formula 1, and the pharmaceutically acceptable salts and solvates thereof (hereinafter xe2x80x9cthe active compoundsxe2x80x9d), may be adminstered through oral, parenteral, topical, or rectal routes in the treatment or prevention of bacterial or protozoa infections. In general, these compounds are most desirably administered in dosages ranging from about 0.2 mg per kg body weight per day (mg/kglday) to about 200 mg/kg/day in single or divided doses (i.e., from 1 to 4 doses per day), although variations will necessarily occur depending upon the species, weight and condition of the subject being treated and the particular route of administration chosen. However, a dosage level that is in the range of about 4 mg/kg/day to about 50 mg/kg/day is most desirably employed. Variations may nevertheless occur depending upon the species of mammal, fish or bird being treated and its individual response to said medicament, as well as on the type of pharmaceutical formulation chosen and the time period and interval at which such administration is carried out In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effects, provided that such larger doses are first divided into several small doses for administration throughout the day.
The active compounds may be administered alone or in combination with pharmaceutically acceptable carriers or diluents by the routes previously indicated, and such administration may be carried out in single or multiple doses. More particularly, the active compounds may be administered in a wide variety of different dosage forms, i.e., they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, oral pharmaceutical compositions can be suitably sweetened and/or flavored. In general, the active compounds are present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by weight
For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (and preferably corm, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the active compound may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
For parenteral administration, solutions of an active compound in either sesame or peanut oil or in aqueous propylene glycol may be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8) if necessary and the liquid diluent first rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques will known to those skilled in the art.
Additionally, it is also possible to administer the active compounds of the present invention topically and this may be done by way of creams, jellies, gels, pastes, patches, ointments and the like, in accordance with standard pharmaceutical practice.
For administration to animals other than humans, such as cattle or domestic animals, the active compounds may be administered in the feed of the animals or orally as a drench composition.
The active compounds may also be adminstered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
The active compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide phenyl, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepollysine substituted with palmitoylresidues. Furthermore, the active compounds may be coupled to a dass of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic add, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric add, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
The Examples provided below illustrate specific embodiments of the invention, but the invention is not limited in scope to the Examples specifically exemplified. 